Previously, live cell chambers such as the Leiden, Sykes-Moore, and Dvorak-Stotler culture chambers were designed primarily for use on an inverted microscope at lower magnifications. These chambers would permit the infusion of liquids onto optical surfaces that due to the presence of a large number of intervening structural components were too far apart from each other and too far away from the stage to obtain the optimum illumination or desired image quality for the most commonly used forms of microscopy such as brightfield, darkfield, phase, DIC, and fluorescence. Another drawback of previous chambers is that the infusion method directed a concentrated stream of liquid across the sample thus washing away the specimen.
As if these problems were not perplexing enough, the older chambers were difficult to load and assemble; not to mention that temperature control was difficult to maintain, and in some cases efforts to control temperature resulted in damage to the microscope optics.
These longstanding problems have continually challenged those people engaged in live cell research. Since nothing else was available on the market that gave the researchers what they wanted, they used whatever was available until the demands of high resolution fluorescent microscopy made it necessary for a new design to handle the requirements of live cell research.
The ideal live cell chamber construction sought by the researchers in fluorescent microscopy would fulfill the following operating parameters: enhanced performance from the microscope; long term life of the cells within a closed system; rapid infusion of the cells into and through the chamber; laminar flow of a preequilibriated medium through the chamber; as well as, a temperature regulation and stabilization of the medium both within the chamber and throughout the closed circuit system.
In addition, all of the structural components of the live cell chamber would have to be nonreactive to biological materials and fit on a standard microscope with an absolute minimum of modification to the original design of the scope. The chamber must also be capable of conducting an ample volume of media in a laminar motion across the specimen without leaking or damaging the cells. The ideal design should further allow for easy care and maintenance of the live cell chamber; as well as, ease of loading of specimens.
From an optical standpoint the live cell chamber must allow the specimen to be viewed using Koehler illumination. The particular spacing and the optical materials employed have to be compatible with standard microscopy; and, the viewing surface has to be large enough to allow room for movement of the chamber when used with large diameter objectives to present the cells in such a way that they can be observed at the highest degree of resolution without damage to the cells.
From a temperature standpoint the live cell chamber temperature has to be able to be regulatable to accommodate the characteristics of different samples. In order to do this the temperature has to be monitored in the media flow region, and once the required temperature has been obtained it also must be stabilized within one tenth of a degree centigrade.